Insulators are genomic elements required for transcriptional fidelity in eukaryotic genomes. Mounting evidence indicates that insulator binding proteins are multi-faceted transcriptional regulators, contributing to insulator, activator and repressor functions. This diversity in action implies that not all binding sites for insulator proteins are bona fide insulators. The challenge in the field is to identify true insulators among thousands of binding sites and define parameters that shape a single DNA binding protein into a multi- functional regulator. The goal of this proposal is to define the function of the prototypic Suppressor of Hairy-wing [Su(Hw)] protein, the DNA binding component of the gypsy insulator. Three aims are proposed. First, we will define the role of the conserved zinc finger Su(Hw) domain in target site selection and partner recruitment. Second, we will define how Su(Hw) contributes to transcriptional regulation. Third, we will determine Su(Hw) function in early development. These studies will enhance our understanding of how a classic insulator binding proteins organizes transcriptional domains throughout the genome. As defects in insulator function are linked to cancers, imprinting syndromes and repeat expansion diseases, our studies will enhance our understanding of how these regulatory elements contribute to nuclear functions important for human health. PUBLIC HEALTH RELEVANCE: Insulators are genomic elements that define domains of independent transcription. Understanding mechanisms of insulator action is critical for improved design of gene therapy vectors, as current approaches face challenges such as inappropriate activation of cellular genes and inadequate long-term expression of therapeutic genes. Our studies will lead to the better selection of insulators for inclusion in the next generation of gene therapy vectors to mitigate unwanted effects on cellular genes and improve long-term of expression of therapeutic genes.